Mullerian Inhibiting Substance (MIS) also known as anti-Mullerian hormone (AMH), is a 140-kDa disulfide-linked homodimer glycoprotein member of the large transforming growth factor-β (TGFβ) multigene family of glycoproteins. The proteins in this gene family are all produced as dimeric precursors and undergo posttranslational processing for activation, requiring cleavage and dissociation to release bioactive C-terminal fragments. Similarly, the 140 kilodalton (kDa) disulfide-linked homodimer of MIS is proteolytically cleaved to generate its active C-terminal fragments.
MIS, is a reproductive hormone produced in fetal testes, which inhibits the development of female secondary sexual structures in males. Before sexual differentiation, the fetus is bipotential, and the developmental choice of male Wolffian ducts (i.e. prostate, vas deferens) over female Mullerian ducts (i.e. Fallopian tubes, uterus, vagina) in the male is controlled in part by MIS.
The human MIS gene is located on chromosome 19, and its expression is sexually dimorphic. In males, MIS expression begins at 9 weeks gestation in the fetal testes and continues at high levels until puberty, when expression levels fall dramatically. In females, MIS is produced only postnatally in granulosa cells from prepuberty through menopause at levels similar to adult males, after which expression ceases. In male fetuses MIS causes regression of the Mullerian ducts, the precursors to the Fallopian tubes, uterus, cervix, and upper third of the vagina.
MIS exerts its biologic effect after binding to a heterodimer of type I and type II single transmembrane spanning serine threonine kinase receptors, leading to cross phosphorylation of the GS box kinase domain of the type I receptor by the type II receptor. Subsequently, SMAD 1, 5 and 8 (but predominantly SMAD 8) are activated and, together with SMAD 4, regulate gene transcription. Only one MIS receptor type II (MISRII) gene has been identified in mice, rats, and rabbits, where in humans its gene localizes to chromosome 12. It is a 65-kDa protein which has been detected in embryonic and adult Mullerian structures, breast tissue, prostatic tissue, the gonads, motor neurons, and brain. In the fetus, mesoepithelial cells expressing MISRII in the coelomic epithelium covering the urogenital ridge migrate into and become part of the mesenchymal cells surrounding the Mullerian duct epithelium. Expression is also detected in the gonads, as wells as in the ovarian coelomic epithelium. Type I MIS receptors have been identified in mammals, with activin receptor-like kinase (ALK) 2 and 3 being the most likely candidates, depending upon animal species and the tissue examined.
In addition to its well established role in the regression of Mullerian ducts, MIS inhibits the proliferation of various human cancer cell lines in vitro and in vivo. The cell lines showing inhibition were derived from ovarian, cervical, endometrial, prostate, uterine, Fallopian and breast cancers. Toxicity has not been observed in vivo even when high concentrations of MIS are maintained systemically in rodents or in human patients with tumors secreting MIS for prolonged periods of time. These findings of relatively restricted receptor expression, anti-proliferative activity against cancer cells expressing the MIS RI and RII, and its apparent non-toxicity, taken together, make MIS an ideal reagent for use in combination with existing chemotherapeutic drugs for the treatment of ovarian cancer, which are known to become resistant to these conventional agents.
MIS acts through MIS Type II receptor cells to serve as a potent tumor suppressor of ovarian cancer initiation (Teixeira et al, unpublished). MIS can also target, as a receptor mediated event the stem/progenitor population of the ovarian cancer cell line (Meirelles et al, 2012; Wei et al, 2010). MIS can be used for the treatment of cancers, for example, expressing MISRII. MISRII is expressed in the majority of epithelial ovarian cancers (Masiakos et al. 1999; Bakkum-Gamez et al. 2008; Song et al. 2009).
MIS also inhibits growth of a variety of cancers in vitro and in vivo, without obvious toxicity after prolonged therapy in vivo (Pieretti-Vanmarcke et al. 2006b). Epithelial ovarian cancer recapitulates the original histology of the embryonic Mullerian ducts and its various subtypes (Scully 1977); for example, serous cystadenocarcinoma resembles embryonic Fallopian tube, endometrioid carcinoma, the endometrium, and mucinous carcinoma, the cervix. Also, MIS acts synergistically or additively with commonly used cancer drugs to control tumor growth (Pieretti-Vanmarcke et al. 2006a).
It has been previously reported that chemotherapeutic agents select for ovarian cancer stem cells, which are typically multi-drug resistant, and/or resistant to chemotherapeutics. In particular, there is a growing body of research reporting that ovarian cancers and cell lines are heterogeneous, with ovarian cancer stem cell populations that are resistant to chemotherapeutic drugs but remain responsive to MIS. MIS particularly targets ovarian cancer side population cells and a population of CD44+, CD24+, EpCam+ and E-Cadherin-negative cells that are ovarian cancer progenitor cells with stem/progenitor characteristics that respond poorly to chemotherapeutic agents currently in clinical use for ovarian cancer (Wei et al, 2010). In particular, MIS has been shown to inhibit ovarian cancer cells both in-vitro and in-vivo and can specifically target and inhibit the growth of an ovarian cancer progenitor cell population enriched by the CD44+, CD24+, Ep-CAM+ and E-cadherin-cell surface markers. In order to accommodate clinical testing of MIS in ovarian cancer patients, the production of recombinant human MIS must be optimized to increase yield and purity.
MIS belongs to the TGF-β superfamily, a class of proteins involved in many pathologies including cancer. Recombinant TGF-β proteins have been very difficult to produce because they require complex maturation process involving pre-pro protein cleavage, dimerization, and glycosylation and disulfide bonding for activity. Previous attempts have been plagued by low production, limited cleavage, and lack of homogeneity, even in mammalian cells. In particular, MIS can only be feasibly produced in mammalian cells, and not E. coli or yeast, where production yields are much higher, and industrial scaling more straightforward. In mammalian cells, yields and homogeneity of the product can be significant barriers to industrial scaling and ultimate entry into clinical trials. For example, proteolytic degradation was a contributing factor to the failure of topical TGF-β3 in early clinical trials against chemotherapy-induced oral mucositis in patients with lymphomas and solid tumors. Recombinant BMP-2 in a paste form remains the only TGF-β family ligand used in the clinic, and is limited to the specific indication of autologous bone grafting. Progress in the technology of production and purification of TGF-β recombinant proteins could help many candidates to achieve their therapeutic potential in the clinic.
Accordingly, the preparation resulting from purification of native or wild-type MIS is complex and the yield is low. Furthermore, the cleavage necessary to produce the active fragment of MIS is also inefficient. Human MIS protein is produced from a pre-proprotein, which comprises a leader sequence. The leader sequence (amino acids 1-25 of SEQ ID NO: 1) is cleaved off and the remaining preprotein (often called “holo-human MIS”) must be post-translationally cleaved to result in a N-terminal and C-terminal domain. These covalently linked N-terminal and C-terminal domains form a monomer, and two identical monomers (comprising the N- and C-terminal domains) form together to generate a homodimer Holo-human MIS is cleaved into its N- and C-terminal domains most likely by means of furin or a related prohormone convertase PC5, expressed in the gonads. Cleavage occurs primarily at a kex-like site characterized by R−4 XXR−1 with a serine in the +1 site, which makes the MIS cleavage site monobasic. The purified C-terminal domain is the biologically active moiety and cleavage is required for biological activity. A secondary cleavage site, whose significance is unknown, is observed less frequently at residues 229-230 (which corresponds to amino acid residues 254-255 of SEQ ID NO:1). Non-cleavable mutants of MIS are not biologically active and mutations in the human gene that truncate the carboxy-terminal domain lead to persistent Mullerian duct syndrome. The role of the amino-terminal domain in vivo may be to assist in protein folding and to facilitate delivery of the C-terminal peptide to its receptor. In one study (Cate, Pepinsky, et al.) addition of the N-terminal peptide was shown to enhance the biological activity of the C-terminal moiety in vitro, but the mechanism was unclear. The cleavage of recombinant MIS expressed by CHO cells is incomplete, thus cleavage with an exogenous serine protease such as plasmin is required to enhance bioactivity.
Accordingly, there is a need for a more efficient method to produce high concentrations of human MIS protein for use as a therapeutic biologic agent.